Synthesis and biological activity of novel 4- and 6-(1-alkyl/aryl-1H- benzimidazol-2-yl)benzene-1,3-diols

Article abstract of DOI:10.1007/s00706-011-0665-5

A one-pot synthesis of new biologically active 4- and 6-(1-alkyl/aryl-17/- benzimidazol-2-yl)benzene-1,3-diols has been developed. The compounds were obtained by the reaction of aryl-modified sulfinylbis[(2,4-dihy-droxyphenyl) methanethione] with N-substituted benzene-1,2-diamines. Elemental analysis, IR, ¹H NMR, ¹³C NMR, and mass spectral data were used to elucidate their structures. The developed method offers short reaction times, easy and quick isolation of the products, and good yields. The antiproliferative properties of the synthesized compounds were investigated against a panel of human cancer cell lines. Some of the tested compounds showed significant cytotoxic activity.

Full text of DOI:10.1007/s00706-011-0665-5

Monatsh Chem (2012) 143:269–276
DOI 10.1007/s00706-011-0665-5
ORIGINAL PAPER
Synthesis and biological activity of novel
4- and 6-(1-alkyl/aryl-1H-benzimidazol-2-yl)benzene-1,3-diols
•
Monika M. Karpinska Joanna Matysiak
•
´
Andrzej Niewiadomy Joanna Wietrzyk
Dagmara Kłopotowska
•
•
Received: 13 April 2011 / Accepted: 25 September 2011 / Published online: 22 October 2011
Ó Springer-Verlag 2011
Abstract A one-pot synthesis of new biologically active
4- and 6-(1-alkyl/aryl-1H-benzimidazol-2-yl)benzene-1,3-
diols has been developed. The compounds were obtained
by the reaction of aryl-modified sulfinylbis[(2,4-dihy-
droxyphenyl)methanethione] with N-substituted benzene-
1,2-diamines. Elemental analysis, IR, 1H NMR, 13C NMR,
and mass spectral data were used to elucidate their struc-
tures. The developed method offers short reaction times,
easy and quick isolation of the products, and good yields.
The antiproliferative properties of the synthesized com-
pounds were investigated against a panel of human cancer
cell lines. Some of the tested compounds showed signifi-
cant cytotoxic activity.
functional materials [13–17]. Therefore, the construction of
these heterocycles has received much attention.
The classical method for the synthesis of benzimidaz-
oles is via the condensation of benzene-1,2-diamines with
carboxylic acids or their derivatives under strong acid
(polyphosphoric acid (PPA) or other mineral acids)/high
temperature conditions [2, 18–20]; another simple and
efficient procedure uses the corresponding aldehydes under
oxidative conditions [21]. Although these transformations
are widely used in the preparation of benzimidazoles, there
remain many drawbacks to overcome such as the use of
highly toxic reagents, strong acids and, in some cases,
harsh reaction conditions [22].
Bahrami et al. [23] described the synthesis of benzimi-
dazoles from classical substrates catalyzed by ceric
ammonium nitrate (CAN) in the presence of H2O2 or
promoted by H2O2/Fe(NO3)3 [24]. Others used a sulfamic
acid/methanol catalytic system in the synthesis [25]. A
highly efficient and versatile method for the synthesis of a
series of 2-substituted N–H-, N-alkyl-, and N-arylbenzim-
idazoles containing a wide range of functional groups was
achieved in one step via the Na2S2O4 reduction of o-ni-
troanilines in the presence of aldehydes [26]. Ridley et al.
[27] and others [28] obtained benzimidazoles by con-
densing diamines with bisulfites in ethanol.
Keywords 4-(1H-Benzimidazol-2-yl)benzene-1,3-diols Á
Antitumor agents Á Antiproliferative activity Á Synthesis
Introduction
Benzimidazoles are frequently found in a diverse array of
compounds, including biologically [1–8] and therapeuti-
cally active [9] agents, natural products [10–12], and
´
M. M. Karpinska Á A. Niewiadomy
The transition-metal-catalyzed C–N cross-coupling
methodology is another approach for the synthesis of
benzimidazole. For example, Brain and Brunton [29]
reported the first palladium-catalyzed N-arylation of
(o-bromophenyl)amidines to give benzimidazoles in tolu-
ene. Evindar and Batey [30] demonstrated an
intramolecular aryl guanidinylation to form biologically
relevant 2-aminobenzimidazoles using both palladium
and copper catalysts in 1,2-dimethoxyethane (DME). Pal-
ladium-catalyzed cascade aryl amination/condensation
Institute of Industrial Organic Chemistry in Warsaw,
Warsaw, Poland
J. Matysiak (&) Á A. Niewiadomy
Department of Chemistry, University of Life Sciences in Lublin,
Akademicka 15, 20-950 Lublin, Poland
e-mail: joanna.matysiak@up.lublin.pl
J. Wietrzyk Á D. Kłopotowska
Department of Experimental Oncology, Institute of Immunology
and Experimental Therapy, Polish Academy of Sciences,
R. Weigla 12, 53-114 Wrocław, Poland
123

´
M. M. Karpinska et al.
270
HO
R1
R2
processes of o-haloacetanilides have been developed for
the synthesis of 1,2-disubstituted benzimidazoles in dime-
thyl sulfoxide (DMSO) [31].
NH2
NH
1: R1 = Me, R2 = H
2: R1 = H, R2 = Et
3: R1 = OH, R2 = H
N
N
OH
CH3
CH3
Recently Li et al. [32] reported a practical, cheap, and
efficient copper-catalyzed intramolecular N-arylation of
(o-haloaryl)amidines to afford synthetically valuable benz-
imidazole derivatives with environmentally benign water as
the solvent. In recent years solvent-free synthesis of benz-
imidazoles under microwave irradiation using Yb(OTf)3
[33], KSF clay [34], PPA, metal halide supported alumina
[35], and other solid supports [36, 37] has been reported.
Nagawade and co-workers described the analogous reaction
using BF3ÁOEt2 and TiCl4 as catalysts [38, 39].
1-3
HO
O2N
NH2
NH
O2N
N
N
4: R = H
OH
5: R = Et
6: R = Cl
OH
R
OH
4-6
HO
R1
7: R1 = R2= H
NH2
NH
N
8: R1 = Me, R2= H
9: R1 = H, R2= Me
10: R1 = H, R2= Et
11: R1 = H, R2= Cl
12: R1 = OH, R2= H
OH
N
R2
In continuation of an extensive program directed toward
the synthesis of biologically active heterocyclic benzen-
ediols, several new benzimidazole derivatives bearing an
alkyl or aryl substituent at the N1 position were designed
and obtained. The antiproliferative potency of the com-
pounds against a panel of human cancer cell lines was
evaluated.
7-12
HO
R1
Cl
NH2
NH
Cl
N
13: R1 = H, R2 = Et
14: R1 = OH, R2 = H
OH
N
R2
COOH
COOH
13, 14
Results and discussion
Scheme 1
Chemistry
OH
R1
S
S
OH
R2
R1
OH
STB: R1 = R2 = H
SClTB: R2= Cl, R1 = H
S3MTB: R1 = Me, R2 = H
S5MTB: R2 = Me, R1 = H
SETB: R2 = Et, R1 = H
S3TTB: R1= OH, R2 = H
Here we report the preparation of novel benzenediole
derivatives containing the highly bioactive benzimidazole
moiety by a new synthesis elaborated by us; however, the
approach is similar to conventional ones. The compounds
were formed by the reaction of N-alkyl(aryl)benzene-1,2-
diamine derivatives with sulfinylbis[(2,4-dihydroxyphenyl)-
methanethione] (STB) or its analogues sulfinylbis[(2,4-
dihydroxy-3-methylphenyl)methanethione] (S3MTB), sul-
finylbis[(2,4-dihydroxy-5-methylphenyl)methanethione]
(S5MTB), sulfinylbis[(5-chloro-2,4-dihydroxyphenyl)me-
thanethione] (SClTB), and sulfinylbis[(2,3,4-trihydroxy-
phenyl)methanethione] (S3TTB) in methanol under reflux
(2.5–3.5 h) in moderate to good yields (63–77%) as out-
lined in Scheme 1. These key derivatives were prepared by
the reaction of 2,4-dihydroxybenzenecarbodithioic acids
with SOCl2 in anhydrous diethyl ether [40]. The diamines
react with the thione electrophiles according to proposed
mechanism in Scheme 2, affording the target compounds
by the elimination of H2S from the imine-thiole adduct.
The reaction proceeds quickly and does not require drastic
conditions, additional reagents, or toxic/hazardous sol-
vents, because STB and its analogs are better electrophiles
than aldehydes, which is the result of the electronic effects
of two OH substituents in the phenyl ring and the SO
group. As a consequence of these interactions and prop-
erties of the solvent, easily outgoing reactive carbocations
are released. The cyclization process promotes a tendency
S
O
HO
R2
HO
OH
H
R
NH2
N
STB
R'
R'
S
NH
R"
NH
R"
HO
OH
HO
R
N
N
N
-H2S
OH
R
R'
R'
SH
NH
R"
R"
R = H, Me, Et, Cl, OH
R' = H, Cl, NO2
R" = Me, HOCH2CH2, Ph, 4-HOOC-C6H4
Scheme 2
of the compounds with a thioamide moiety toward transi-
tion into the thiol-imine forms compared with the analogs
with an O atom.
Purity of the compounds was monitored by reversed-
phase (RP-18) HPLC chromatography with methanol–
water as a mobile phase at pH 4. Log k values for 70%
MeOH (v/v) in the mobile phase are presented. The
structures of compounds under consideration were con-
firmed by the analytical and spectral data. The mass spectra
123

Synthesis and biological activity
271
(EI) of compounds gave molecular ion peaks, albeit with
different intensities. The IR spectra showed an intense band
in the region of about 1,630 cm-1 corresponding to the
vibration of C=N which confirmed the formation of
the desired compounds. Further, there are characteristic
Table 2 Antiproliferative potency of some compounds against the
human cancer cell lines T47D, A549, and SW707 expressed as IC50
No.
IC50/lM
A549
T47D
SW707
v (O–H) vibration frequencies between 3,350 and 3,550 cm-1
.
7
28.9 1.1***
15.0 2.2
17.1 7.1
9.7 1.7
18.5 2.4*/***
12.7 2.2
60.0 13.5***
23.5 6.4
In the 1H NMR spectra the –OH groups are detected as broad
singlets at about 10 and 12–12.9 ppm [41]; in the case of
compounds 2, 10, and 14, these signals were not detected.
8
9
12.1 1.7
34.0 17.1
10.7 2.2
10
8.0 2.2**
10.1 2.5**
17.6 5.4
11
12.5 1.0
6.8 5.4
12.1 0.8
Antiproliferative activity
Cisplatin
10.4 5.0****
Statistically significant differences of the compounds’ potency
between *T47D and SW707, **T47D and HCV29T cell lines; sta-
tistically significant differences of the cell line sensitivity to
compounds ***7 and 10, ****7 and cisplatin; (p \ 0.05; Kruskal–
Wallis ANOVA test)
The 4-(1H-benzimidazol-2-yl)benzene-1,3-diols presented
in Scheme 1 were evaluated for their antiproliferative
potency against human immortalized tumorigenic HCV29T
cells. The cytotoxic activity in vitro was expressed as IC50,
the concentration of the compound that inhibited prolifera-
tion rate of the tumour cells by 50% as compared to the
untreated control cells. Cisplatin was used as a reference
drug. The results of the screening are summarized in Table 1.
The results showed that most of our designed compounds
possess good to moderate antiproliferative potency with IC50
values between 17.0 and 171.9 lM (Table 1). Potency of the
compounds depends clearly on the type of substitution of
both rings. Generally (1-phenyl-1H-benzimidazol-2-
yl)benzene-1,3-diols 7–11 and compound 13 show the
strongest antiproliferative effect. Of the compounds with an
unmodified resorcyl moiety (4, 7) a significantly higher
effect against HCV29T cells is exhibited by compound 7
which has an N-phenyl substituent (Table 1). A similar trend
is observed in the group of compounds with an ethyl sub-
stituent (compounds 2, 5, 10, 13). Derivatives with a third
hydroxyl substituent possess remarkably lower antiprolif-
erative potency in the individual groups (3, 12, 14).
Compounds 8, 9, 10, and 11 exhibit similar antiprolif-
erative potency compared to that of cisplatin (p = 0.05).
The structure–activity (SAR) analysis shows that the
additional hydrophobic substituent in the resorcinol ring
increases antiproliferative potency. The compounds with
an ethyl substituent display higher antiproliferative
potency. The additional hydroxyl group decreases biolog-
ical activity. This trend is similar to that observed for 4H-
3,1-benzothiazines obtained by us [42]. At the same time
these groups of derivatives confirm and extend the earlier
finding that the presence of a chlorine atom or methyl (or
ethyl, isopropyl) substituents in position 5 of the resorcinol
moiety of resorcyl(benz)azoles improves their anticancer
properties [43, 44].
Conclusions
Selected compounds of the highest potency against
HCV29T cells were also tested against A549 (human non-
small lung carcinoma), T47D (human breast cancer), and
SW707 (human rectal adenocarcinoma) cells (Table 2).
We designed a new synthesis of 1-substituted-1H-benz-
imidazoles. This approach offers short reaction times,
relatively large-scale synthesis, easy and quick isolation of
the products, and good to moderate yields. 4-(1H-Benz-
imidazol-2-yl)benzene-1,3-diols obtained in this way were
evaluated for their antiproliferative activities. All com-
pounds demonstrated potent inhibition against all the
human cancer cell lines tested. Furthermore, we also con-
clude that compound 10 showed similar antiproliferative
potency to the standard cisplatin.
Table 1 Antiproliferative potency of 4-(1H-benzimidazol-2-yl)ben-
zene-1,3-diols against the human cancer cell line HCV29T expressed
as IC50
No.
IC50/lM
No.
IC50/lM
1
2
3
4
5
6
7
8
113.1 12.4
81.8 13.9
160.8 22.3
171.9 20.5
59.6 8.4
9
35.7 11.2
17.0 2.5
21.9 0.4
46.2 11.7
24.9 10.1
95.6 9.8
10
11
12
13
14
Experimental
55.2 17.5
48.6 12.4
25.8 6.6
General
Cisplatin
3.1 1.0
¨
Melting points were determined using a BUCHI B-540
(Switzerland) melting point apparatus. Elemental analyses
IC50 values are the means SD of 9 independent experiments
123

´
M. M. Karpinska et al.
272
4-Ethyl-6-(1-methyl-1H-benzimidazol-2-yl)benzene-1,3-
diol (2, C16H16N2O2)
(C, H, N) were conducted using a Perkin-Elmer 2400
instrument and were found to be in good agreement
( 0.2%) with the calculated values. The IR spectra were
recorded with a Perkin-Elmer FT-IR 1725X spectropho-
tometer (in KBr). The spectra were measured in the range
A
mixture of 0.24 g N-methylbenzene-1,2-diamine
(2 mmol) and 0.74 g SETB (2 mmol) in 16 cm3 MeOH
was refluxed for 3.5 h. The hot mixture was filtered and the
filtrate was concentrated. The obtained solid was combined
with that formed after reconcentration of the filtrate.
Recrystallization from MeOH/H2O (2:1) afforded 0.41 g
1
of 600–4,000 cm-1. H NMR and 13C NMR spectra were
recorded in DMSO-d6 by means of a Bruker DRX 500
instrument. Chemical shifts (d, ppm) were given in relation
to tetramethylsilane (TMS). Mass spectra (EI, 70 eV) were
recorded using the apparatus AMD-604.
ꢀ
(76%) 2. M.p.: 213–216 °C; IR (KBr): m = 3,394 (OH),
3,243 (OH), 1,638 (C=N), 1,520 (C=C), 1,502 (C=C),
1,463, 1,307, 1,224 (C–OH), 1,186, 1,148, 1,077, 1,054,
993, 945, 927, 862, 802, 756, 713 cm-1 1H NMR
;
The purity of the compounds was examined by a Knauer
liquid chromatograph equipped with a dual pump, a
20-mm3 simple injection valve, and a UV–Vis detector at
280 nm. A Hypersil Gold C18 (3 lm, 100 9 3 mm) col-
umn was used as the stationary phase. The mobile phase
included different contents of MeOH and acetate buffer
(pH 4, 20 nM) as the aqueous phase. The flow rate was
0.5 cm3 min-1 at room temperature. The retention time of
an unretained solute (to) was determined by the injection of
a small amount of acetone dissolved in water. Log k values
for 70% MeOH (v/v) in the mobile phase are presented.
Log k values are calculated as log k = log (tR - to)/to,
where tR = retention time of a solute, to = retention time
of an unretained solute.
(500 MHz, DMSO-d6): d = 10.20 (s, OH), 7.84 (d,
J = 7.4 Hz, 1H, Ar–H), 7.74 (m, 1H, Ar–H), 7.49 (m,
2H, Ar–H), 7.36 (s, 1H, Ar–H), 6.65 (s, 1H, Ar–H), 3.89 (s,
CH3), 2.54 (q, J = 7.5 Hz, CH2), 1.16 (t, J = 7.5 Hz,
CH3) ppm; 13C NMR (125 MHz, DMSO-d6): d = 161.2,
155.8, 150.2, 133.6, 131.1, 128.6, 124.9, 125.1, 114.2,
112.6, 112.8, 108.1, 102.0, 33.8 (CH3), 21.6 (CH2), 14.8
(CH3) ppm; MS (70 eV): m/z = 268 (M?, 25), 253
(M? - CH3, 100), 239 (6), 183 (6), 168 (3), 164 (7), 159
(12), 151 (6), 123 (5), 119 (3), 77 (9), 51 (4), 44 (5), 39 (4);
HPLC (C-18): log k = -0.829.
4-(1-Methyl-1H-benzimidazol-2-yl)benzene-1,2,3-triol
(3, C14H12N2O3)
Starting materials were purchased from Aldrich (N-
methylbenzene-1,2-diamine, 2-[(2-amino-4-nitrophenyl)
amino]ethanol, 4-[(2-amino-4-chlorophenyl)amino]benzoic
acid) or Alfa Aesar (N-phenylbenzene-1,2-diamine).
A
mixture of 0.24 g N-methylbenzene-1,2-diamine
(2 mmol) and 0.71 g S3TTB (2 mmol) in 16 cm3 MeOH
was refluxed for 4 h. The hot mixture was filtered and the
filtrate was concentrated. Recrystallization from MeOH/
H2O (3:2) afforded 0.29 g (63%) 3. M.p.: [410 °C; IR
2-Methyl-4-(1-methyl-1H-benzimidazol-2-yl)benzene-1,3-
diol (1, C15H14N2O2)
ꢀ
(KBr): m = 3,146 (OH), 2,935 (CH), 1,618 (C=N), 1,567
A
mixture of 0.24 g N-methylbenzene-1,2-diamine
(2 mmol) and 0.74 g S3MTB (2 mmol) in 18 cm3 MeOH
was refluxed for 3 h. The hot mixture was filtered. The
obtained solid was combined with that formed after the
concentration of the filtrate. Recrystallization from meth-
anol afforded 0.35 g (68%) 1. M.p.: 295–298 °C; IR
(KBr): ꢀm = 3,145 (OH), 2,915 (CH), 2,860 (CH), 1,612
(C=N), 1,562 (C=C), 1,516 (C=C), 1,485, 1,458, 1,407,
1,359, 1,313, 1,240, 1,223 (C–OH), 1,155, 1,076, 1,010,
(C=C), 1,509 (C=C), 1,472, 1,394, 1,309, 1,273, 1,208
(C–OH), 1,077, 1,035, 1,003, 938, 916, 869, 811, 758, 736,
1
708 cm-1; H NMR (500 MHz, DMSO-d6): d = 10.72 (s,
OH), 9.62 (s, OH), 8.51 (s, OH), 7.65 (m, 2H, Ar–H), 7.29 (m,
2H, Ar–H), 7.14 (d, J = 8.6 Hz, 1H, Ar–H), 6.49 (d,
J = 8.6 Hz, 1H, Ar–H), 3.91 (s, CH3) ppm; 13C NMR
(125 MHz, DMSO-d6): d = 162.2, 155.6, 159.8, 146.9,
135.5, 129.0, 124.9, 125.1, 117.2, 116.6, 112.2, 110.1, 104.1,
38.4 (CH3) ppm; MS (70 eV): m/z = 256 (M?, 100), 255
(70), 239 (52), 227 (21), 200 (13), 184 (19), 168 (18), 157 (9),
152 (63), 131 (6), 124 (15), 106 (8), 77 (12), 68 (8), 44 (21),
39 (13), 36 (11); HPLC (C-18): log k = -0.709.
938, 905, 875, 821, 797, 751, 719 cm-1
;
1H NMR
(500 MHz, DMSO-d6): d = 10.40 (s, OH), 9.88 (s, OH),
7.97 (dd, J = 6.9, 1.9 Hz, 1H, Ar–H), 7.81 (dd, J = 7.0,
2.0 Hz, 1H, Ar–H), 7.60 (m, 2H, Ar–H), 7.32 (d,
J = 8.5 Hz, 1H, Ar–H), 6.72 (d, J = 8.5 Hz, 1H, Ar–H),
3.87 (s, CH3), 2.12 (s, CH3) ppm; 13C NMR (125 MHz,
DMSO-d6): d = 160.8, 155.4, 149.9, 132.9, 131.0, 129.0,
125.8, 125.2, 114.0, 112.8, 112.7, 108.0, 101.9, 32.4 (CH3),
9.1 (CH3) ppm; MS (70 eV): m/z = 254 (M?, 100), 237
(46), 225 (15), 222 (4), 206 (3), 197 (4), 183 (4), 171 (3),
157 (2), 146 (2), 127 (2), 104 (2), 91 (2), 77 (6), 65 (3), 51
(3), 36 (7); HPLC (C-18): log k = -0.274.
4-[1-(2-Hydroxyethyl)-5-nitro-1H-benzimidazol-2-yl]-
benzene-1,3-diol (4, C15H13N3O5)
A mixture of 0.26 g 2-[(2-amino-4-nitrophenyl)amino]eth-
anol (1.3 mmol) and 0.46 g STB (1.3 mmol) in 10 cm3
MeOH was refluxed for 4 h. The hot mixture was filtered
and the filtrate was concentrated. Recrystallization from
methanol afforded 0.32 g (77%) 4. M.p.: 279–282 °C; IR
ꢀ
(KBr): m = 3,326 (OH), 3,133 (OH), 1,610 (C=N), 1,599
123

Synthesis and biological activity
273
(C=N), 1,536 (C=C), 1,487 (C=C), 1,433, 1,357 (NO2),
1,320, 1,299, 1,253, 1,236 (C–OH), 1,178, 1,124, 1,104,
1,065, 1,040, 998, 982, 958, 926, 900, 866 (NO2), 819,
779, 749, 722 cm-1
;
1H NMR (500 MHz, DMSO-d6):
d = 11.52 (s, OH), 10.87 (s, OH), 8.07 (dd, J = 9.2,
2.5 Hz, 1H, Ar–H), 8.03 (d, J = 2.5 Hz, 1H, Ar–H), 8.00
(s, 1H, Ar–H), 6.88 (d, J = 9.3 Hz, 1H, Ar–H), 6.63 (s, 1H,
Ar–H), 6.47 (s, OH), 3.62 (t, J = 5.6 Hz, OCH2), 3.56 (t,
J = 5.6 Hz, CH2) ppm; 13C NMR (125 MHz, DMSO-d6):
d = 195.3, 156.7, 155.4, 149.8, 135.1, 132.6, 125.3, 125.1,
123.9, 118.7, 111.1, 109.8, 103.9, 59.3 (CH2), 45.2 (CH2)
ppm; MS (70 eV): m/z = 349 (M?, 19), 334 (6), 332 (53),
304 (44), 305 (100), 292 (12), 286 (17), 276 (25), 259 (30),
241 (9), 224 (6), 214 (5), 201 (4), 186 (5), 169 (7), 167 (5),
140 (6), 108 (4), 90 (7), 75 (15), 63 (30), 51 (8), 36 (21);
HPLC (C-18): log k = -0.631.
1
749 cm-1; H NMR (500 MHz, DMSO-d6): d = 11.23 (s,
OH), 10.35 (s, OH), 8.61 (d, J = 7.1 Hz, 1H, Ar–H), 8.36
(dd, J = 9.1, 2.2 Hz, 1H, Ar–H), 8.14 (d, J = 9.1 Hz, 1H,
Ar–H), 7.49 (d, J = 8.5 Hz, 1H, Ar–H), 6.53 (d,
J = 2.3 Hz, 1H, Ar–H), 6.47 (dd, J = 8.5, 2.3 Hz, 1H,
Ar–H), 5.02 (br s, 1H, OH), 4.49 (t, J = 5.3 Hz, OCH2),
3.68 (t, J = 5.3 Hz, CH2) ppm; 13C NMR (125 MHz,
DMSO-d6): d = 161.8, 157.3, 149.9, 135.0, 133.2, 125.2,
125.1, 124.0, 117.4, 109.7, 108.1, 107.5, 102.7, 59.2 (CH2),
45.1 (CH2) ppm; MS (70 eV): m/z = 315 (M?, 19), 298
(20), 284 (4), 272 (20), 271 (100), 252 (10), 241 (6), 238
(6), 225 (17), 168 (2), 90 (3), 76 (6), 63 (4), 52 (2); HPLC
(C-18): log k = -0.760.
4-(1-Phenyl-1H-benzimidazol-2-yl)benzene-1,3-diol
(7, C19H14N2O2)
A
mixture of 0.50 g N-phenylbenzene-1,2-diamine
4-Ethyl-6-[1-(2-hydroxyethyl)-5-nitro-1H-benzimidazol-2-
yl]benzene-1,3-diol (5, C17H17N3O5)
(2.7 mmol) and 1 g STB (3.1 mmol) in 13.5 cm3 MeOH
was refluxed for 3 h. The hot mixture was filtered and the
filtrate was concentrated. NaOH solution (5%) was added
(5 cm3) and the mixture was filtrated. HCl solution (5%)
was added to the filtrate to afford the product. Recrystal-
lization from methanol afforded 0.58 g (71%) 7. M.p.:
A mixture of 0.26 g 2-[(2-amino-4-nitrophenyl)amino]eth-
anol (1.3 mmol) and 0.50 g SETB (1.3 mmol) in 10 cm3
MeOH was refluxed for 3 h. The hot mixture was filtered,
the filtrate was concentrated, and 10 cm3 diethyl ether was
added. Recrystallization from methanol afforded 0.32 g
ꢀ
128–131 °C; IR (KBr): m = 3,438 (OH), 2,921 (CH), 2,849
(CH), 1,612 (C=N), 1,499 (C=C), 1,469, 1,387, 1,323,
ꢀ
(72%) 5. M.p.: 156–159 °C; IR (KBr): m = 3,419 (OH),
2,962 (CH), 2,931 (CH), 2,872 (CH), 1,661 (C=N), 1,620
(C=N), 1,504 (C=C), 1,438, 1,351 (NO2), 1,208 (C–OH),
1,142, 1,087, 1,016, 957, 902, 841 (NO2), 790, 764,
1,288, 1,229 (C–OH), 1,155, 999, 942, 910, 845, 796, 760,
1
725 cm-1; H NMR (500 MHz, DMSO-d6): d = 11.37 (s,
OH), 10.02 (s, OH), 7.81 (d, J = 8.0 Hz, 1H, Ar–H), 7.64
(m, 3H, Ar–H), 7.56 (dd, J = 8.1, 2.0 Hz, 2H, Ar–H), 7.40
(t, J = 7.4 Hz, 1H, Ar–H), 7.34 (t, J = 7.7 Hz, 1H, Ar–H),
7.20 (d, J = 8.4 Hz, 1H, Ar–H), 7.15 (d, J = 7.9 Hz, 1H,
Ar–H), 6.39 (d, J = 2.3 Hz, 1H, Ar–H), 6.12 (dd, J = 8.7,
2.2 Hz, 1H, Ar–H) ppm; 13C NMR (125 MHz, DMSO-d6):
d = 161.3, 160.0, 150.8, 143.8, 136.1, 135.4, 130.4, 130.0,
129.8, 129.2, 127.5, 124.0, 123.9, 117.3, 117.2, 110.7,
107.2, 103.7, 103.4 ppm; MS (70 eV): m/z = 302 (M?,
94), 301 (100), 285 (26), 273 (6), 255 (6), 245 (6), 231 (5),
219 (3), 167 (7), 151 (6), 137 (11), 115 (6), 92 (5), 77 (16),
63 (6), 51 (15); HPLC (C-18): log k = 0.023.
1
735 cm-1; H NMR (500 MHz, DMSO-d6): d = 10.99 (s,
OH), 10.45 (s, OH), 8.56 (d, J = 2.2 Hz, 1H, Ar–H), 8.39
(dd, J = 9.1, 2.2 Hz, 1H, Ar–H), 8.19 (d, J = 9.1 Hz, 1H,
Ar–H), 7.40 (s, 1H Ar–H,), 6.75 (s, 1H, Ar–H), 6.40 (br s,
1H, OH), 4.53 (t, J = 5.3 Hz, OCH2), 3.70 (t, J = 5.3 Hz,
CH2), 2.47 (q, J = 7.4 Hz, CH2), 1.15 (t, J = 7.4 Hz,
CH3) ppm; 13C NMR (125 MHz, DMSO-d6): d = 163.1,
160.9, 155.8, 154.1, 144.8, 137.0, 131.4, 127.8, 120.0,
122.7, 114.6, 110.7, 102.9, 59.2 (CH2), 46.1 (CH2), 22.0
(CH2), 14.0 (CH3) ppm; MS (70 eV): m/z = 343 (M?, 29),
328 (M-CH3, 14), 326 (22), 299 (74), 284 (100), 266 (4),
254 (6), 238 (26), 224 (3), 210 (4), 196 (3), 181 (6), 165
(4), 148 (2), 126 (2), 103 (2), 91 (5), 76 (6), 63 (5), 44 (3),
36 (8); HPLC (C-18): log k = -0.045.
2-Methyl-4-(1-phenyl-1H-benzimidazol-2-yl)benzene-1,3-
diol (8, C20H16N2O2)
A
mixture of 0.26 g N-phenylbenzene-1,2-diamine
4-Chloro-6-[1-(2-hydroxyethyl)-5-nitro-1H-benzimidazol-
2-yl]benzene-1,3-diol (6, C15H12CIN3O5)
(1.4 mmol) and 0.51 g S3MTB (1.4 mmol) in 13.5 cm3
MeOH was refluxed for 3 h. The hot mixture was filtered
and the filtrate was concentrated. Recrystallization from
MeOH/H2O afforded 0.29 g (66%) 8. M.p.: 130–133 °C;
A mixture of 0.26 g 2-[(2-amino-4-nitrophenyl)amino]eth-
anol (1.3 mmol) and 0.51 g SClTB (1.3 mmol) in 10 cm3
MeOH was refluxed for 3 h. The hot mixture was filtered,
the filtrate was concentrated, and 5 cm3 water was added.
Recrystallization from methanol afforded 0.32 g (70%) 6.
ꢀ
IR (KBr): m = 3,062 (OH), 1,671 (C=N), 1,607 (C=C),
1,499 (C=C), 1,455, 1,376, 1,307, 1,266, 1,207 (C–OH),
1,121, 1,081, 1,015, 880, 849, 805, 781, 759, 746 cm-1; 1H
NMR (500 MHz, DMSO-d6): d = 10.75 (s, OH), 10.10 (s,
OH), 7.85 (d, J = 8.0 Hz, 1H, Ar–H), 7.66 (m, 2H, Ar–H),
7.56 (dd, J = 7.8, 2.2 Hz, 2H, Ar–H), 7.51 (t, J = 7.2 Hz,
1H, Ar–H), 7.43 (t, J = 7.5 Hz, 1H, Ar–H), 7.25 (m, 1H,
ꢀ
M.p.: [291 °C (dec.); IR (KBr): m = 3,351 (OH), 3,271
(OH), 2,925 (CH), 1,607 (C=N), 1,551 (C=C), 1,487,
1,441, 1,379 (NO2), 1,285, 1,252 (C–OH), 1,181, 1,150,
1,105 (C–Cl), 1,047, 976, 931, 908, 874, 842 (NO2), 819,
123

´
M. M. Karpinska et al.
274
Ar–H), 7.07 (d, J = 8.0 Hz, 1H, Ar–H), 6.83 (s, 1H, Ar–
H), 6.83 (s, 1H, Ar–H), 2.02 (s, CH3) ppm; 13C NMR
(125 MHz, DMSO-d6): d = 161.4, 160.0, 156.5, 150.0,
134.4, 134.1, 131.1, 130.0, 129.0, 128.0, 127.4, 125.4,
124.9, 117.4, 115.2, 112.2, 111.9, 107.3, 102.2, 8.6 (CH3)
ppm; MS (70 eV): m/z = 316 (M?, 100), 315 (83), 289
(28), 287 (5), 243 (6), 219 (3), 209 (2), 167 (8), 158 (3),
140 (3), 135 (3), 115 (3), 92 (3), 77 (9), 65 (4), 51 (7);
HPLC (C-18): log k = 0.493.
219 (4), 180 (3), 167 (7), 149 (3), 115 (2), 92 (2), 77 (7), 69
(4), 51 (5), 36 (6); HPLC (C-18); log k = 0.567.
4-Chloro-6-(1-phenyl-1H-benzimidazol-2-yl)benzene-1,3-
diol (11, C19H13ClN2O2)
A mixture of 0.26 g N-phenylbenzene-1,2-diamine (1.4
mmol) and 0.54 g SClTB (1.4 mmol) in 13.5 cm3 MeOH
was refluxed for 3 h. The hot mixture was filtered and the
filtrate was concentrated. Recrystallization from MeOH/
H2O (3:2) afforded 0.54 g (71%) 11. M.p.: 133–136 °C; IR
4-Methyl-6-(1-phenyl-1H-benzimidazol-2-yl)benzene-1,3-
diol (9, C20H16N2O2)
ꢀ
(KBr): m = 3,061 (OH, CH), 1,608 (C=N), 1,500 (C=C),
1,452, 1,415, 1,386, 1,299, 1,247, 1,206 (C–OH), 1,106,
1,044, 1,027, 1,006, 981, 925, 884, 839, 749 cm-1; 1H NMR
(500 MHz, DMSO-d6): d = 11.11 (s, OH), 9.98 (s, OH),
7.89 (d, J = 8.0 Hz, 1H, Ar–H), 7.65 (m, 3H, Ar–H), 7.56
(dd, J = 8.1, 2.2 Hz, 2H, Ar–H), 7.53 (m, 1H, Ar–H), 7.47 (t,
J = 7.3 Hz, 1H, Ar–H), 7.32 (d, J = 8.1 Hz, 1H, Ar–H),
7.15 (s, 1H, Ar–H), 6.75 (s, 1H, Ar–H) ppm; 13C NMR
(125 MHz, DMSO-d6): d = 160.2, 157.6, 157.2, 148.6,
134.7, 134.6, 134.3, 130.7, 130.2, 130.0, 127.2, 125.3, 125.2,
116.1, 111.6, 110.6, 103.7, 103.9, 103.2 ppm; MS (70 eV):
m/z = 336 (M?, 100), 319 (24), 301 (10), 271 (5), 255 (4),
243 (14), 231 (6), 219 (4), 205 (3), 194 (3), 171 (7), 166 (5),
140 (5), 136 (5), 122 (6), 102 (3), 91 (2), 76 (3), 63 (4), 51
(14), 36 (10); HPLC (C-18): log k = 0.426.
A
mixture of 0.26 g N-phenylbenzene-1,2-diamine
(1.4 mmol) and 0.51 g S5MTB (1.4 mmol) in 7 cm3 MeOH
was refluxed for 2.5 h. The hot mixture was filtered and the
filtrate was concentrated. Recrystallization from MeOH/
H2O (3:1) afforded 0.30 g (68%) 9. M.p.: 158–159 °C; IR
ꢀ
(KBr): m = 3,400 (OH), 3,056 (CH), 2,919 (CH), 1,625
(C=N), 1,594 (C=C), 1,499 (C=C), 1,453, 1,390, 1,265,
1,255 (C–OH), 1,161, 1,135, 1,071, 1,009, 845, 746,
1
695 cm-1; H NMR (500 MHz, DMSO-d6): d = 10.60 (s,
OH), 10.05 (s, OH), 7.76 (d, J = 7.9 Hz, 1H, Ar–H), 7.66
(m, 2H, Ar–H), 7.51 (dd, J = 8.0, 2.1 Hz, 2H, Ar–H), 7.34
(m, 1H, Ar–H), 7.25 (m, 2H, Ar–H), 7.07 (d, J = 8.0 Hz, 1H,
Ar–H), 6.56 (s, 1H, Ar–H), 6.43 (s, 1H, Ar–H), 2.05 (s, CH3)
ppm; 13C NMR (125 MHz, DMSO-d6): d = 164.0, 158.5,
158.2, 151.3, 136.7, 135.9, 130.2, 129.6, 129.4, 128.9, 127.7,
123.2, 123.0, 117.7, 114.5, 114.4, 110.5, 104.0, 102.7, 15.4
(CH3) ppm; MS (70 eV): m/z = 316 (M?, 100), 299 (23),
287 (13), 271 (5), 259 (6), 245 (4), 231 (5), 219 (3), 206 (3),
167 (9), 151 (4), 139 (3), 115 (4), 103 (2), 92 (3), 77 (10), 65
(4), 51 (8), 39 (4); HPLC (C-18): log k = 0.384.
4-(1-Phenyl-1H-benzimidazol-2-yl)benzene-1,2,3-triol
(12, C19H14N2O3)
A
mixture of 0.26 g N-phenylbenzene-1,2-diamine
(1.4 mmol) and 0.52 g S3TTB (1.4 mmol) in 7 cm3 MeOH
was refluxed for 3.5 h. The hot mixture was filtered and the
filtrate was concentrated. Recrystallization from MeOH/
H2O (3:1) afforded 0.29 g (66%) 12. M.p.: 163–166 °C; IR
4-Ethyl-6-(1-phenyl-1H-benzimidazol-2-yl)benzene-1,3-
diol (10, C21H18N2O2)
ꢀ
(KBr): m = 3,063 (OH), 2,962 (CH), 1,672 (C=N), 1,617
(C=N), 1,551 (C=C), 1,500 (C=C), 1,446, 1,415, 1,388,
A
mixture of 0.26 g N-phenylbenzene-1,2-diamine
1,320, 1,245 (C–OH), 1,149, 1,082, 1,009, 980, 924, 897,
845, 749 cm-1
(1.4 mmol) and 0.54 g SETB (1.4 mmol) in 7 cm3 MeOH
was refluxed for 3.5 h. The hot mixture was filtered and the
filtrate was concentrated. Recrystallization from MeOH/
H2O (3:2) afforded 0.36 g (77%) 10. M.p.: 189–191 °C; IR
;
1H NMR (500 MHz, DMSO-d6):
d = 10.60 (s, OH), 9.98 (s, OH), 9.01 (s, OH), 7.89 (d,
J = 8.1 Hz, 1H, Ar–H), 7.65 (m, 2H, Ar–H), 7.53 (dd,
J = 8.0, 2.3 Hz, 2H, Ar–H), 7.54 (m, 1H, Ar–H), 7.51 (m,
1H, Ar–H), 7.45 (t, J = 7.3 Hz, 1H, Ar–H), 7.28 (d,
J = 8.7 Hz, 1H, Ar–H), 6.48 (d, J = 8.7 Hz, 1H, Ar–H),
6.28 (d, J = 8.7 Hz, 1H, Ar–H) ppm; 13C NMR
(125 MHz, DMSO-d6): d = 152.3, 151.4, 149.9, 149.8,
147.4, 134.7, 134.2, 133.6, 130.3, 130.0, 127.4, 125.4,
125.3, 120.6, 115.6, 111.8, 108.0, 107.8, 102.7 ppm; MS
(70 eV): m/z = 318 (M?, 100), 289 (6), 271 (9), 261 (8),
243 (9), 232 (4), 219 (5), 200 (11), 194 (7), 162 (14), 152
(5), 122 (3), 51 (7), 36 (9); HPLC (C-18): log k = -0.781.
ꢀ
(KBr): m = 3,063 (OH), 2,962 (CH), 1,672 (C=N), 1,617
(C=N), 1,551 (C=C), 1,500 (C=C), 1,446, 1,415, 1,388,
1,320, 1,245 (C–OH), 1,149, 1,082, 1,009, 980, 924, 897,
845, 749 cm-1
;
1H NMR (500 MHz, DMSO-d6):
d = 10.37 (s, OH), 7.98 (d, J = 8.1 Hz, 1H, Ar–H),
7.66–7.64 (m, 3H, Ar–H), 7.57 (m, 2H, Ar–H), 7.53 (m,
1H, Ar–H), 7.48 (t, J = 7.9 Hz, 1H, Ar–H), 7.31 (d,
J = 8.2 Hz, 1H, Ar–H), 6.83 (s, 1H, Ar–H), 6.63 (s, 1H,
Ar–H), 2.24 (q, J = 7.5 Hz, CH2), 0.81 (t, J = 7.5 Hz,
CH3) ppm; 13C NMR (125 MHz, DMSO-d6): d = 163.2,
161.6, 157.3, 149.2, 134.7, 133.8, 132.7, 130.3, 130.1,
130.0, 127.4, 125.6, 125.4, 123.1, 121.8, 115.1, 111.8,
102.7, 100.2, 21.4 (CH2), 13.3 (CH3) ppm; MS (70 eV):
m/z = 330 (M?, 51), 315 (M?-CH3, 100), 301 (5), 243 (5),
4-[5-Chloro-2-(5-ethyl-2,4-dihydroxyphenyl)-1H-benz-
imidazol-1-yl]benzoic acid (13, C22H17ClN2O4)
A mixture of 0.26 g 4-[(2-amino-4-chlorophenyl)amino]
benzoic acid (1 mmol) and 0.52 g SETB (1 mmol) in
123

Synthesis and biological activity
275
5 cm3 MeOH was refluxed for 3.5 h. The hot mixture was
filtered and the filtrate was concentrated. Recrystallization
from methanol afforded 0.27 g (67%) 13. M.p.: 130–
133 °C; IR (KBr): ꢀm = 3,107 (OH), 2,936 (CH), 1,698
(C=O), 1,618 (C=N), 1,503 (C=C), 1,446, 1,406, 1,242
(C–OH), 1,141, 1,098, 1,075, 980, 930, 892, 853, 798, 761,
Twenty-four hours before the addition of the tested agents,
the cells were plated in 96-welI plates (Sarstedt, USA) at a
density of 104 cells/well. All cell lines were maintained in
the opti-MEM medium supplement with 2 mM glutamine
(Gibco, Warsaw, Poland), streptomycin (50 lg cm-3),
penicillin (50 U cm-3) (Polfa, Tarchomin, Poland), and
5% fetal calf serum (Gibco, Grand Island, USA). The cells
were incubated at 37 °C in humid atmosphere saturated
1
704 cm-1; H NMR (500 MHz, DMSO-d6): d = 11.85 (s,
OH), 10.71 (s, COOH), 10.05 (s, OH), 8.13 (dd, J = 7.9,
1.5 Hz, 1H, Ar–H), 7.98 (d, J = 1.9 Hz, 1H, Ar–H), 7.92
(m, 1H, Ar–H), 7.86–7.80 (m, 2H, Ar–H), 7.50 (d,
J = 8.7 Hz, 1H, Ar–H), 7.20 (s, 1H, Ar–H), 6.84 (d,
J = 8.7 Hz, 1H, Ar–H), 6.43 (s, 1H, Ar–H), 2.50 (q,
J = 7.5 Hz, CH2), 1.10 (t, J = 7.5 Hz, CH3) ppm; 13C
NMR (125 MHz, DMSO-d6): d = 169.7 (C=O), 165.0,
163.1, 161.5, 157.7, 134.4, 133.0, 131.7, 131.2, 130.1,
129.3, 127.8, 125.7, 123.1, 120.0, 114.4, 113.8, 113.3,
103.0, 102.5, 21.7 (CH2), 13.0 (CH3) ppm; MS (70 eV):
m/z = 408 (M?, 100), 396 (40), 381 (7), 316 (6), 301 (20),
259 (11), 243 (12), 224 (10), 212 (9), 155 (35), 149 (14),
107 (7), 77 (9), 36 (20); ESI–MS: m/z = 409.1
([M ? H]?); HPLC (C-18): log k = 0.247.
with 5% CO2. The solutions of compounds (1 mg cm-3
)
were prepared ex tempore by dissolving the substance in
100 mm3 of DMSO completed with 900 mm3 of tissue
culture medium. Afterwards, the compounds were diluted
in the culture medium to reach the final concentrations
ranging from 0.1 to 100 lg cm-3. The solvent (DMSO) in
the highest concentration used in the test did not reveal any
cytotoxic activity. Cisplatin was applied as a test reference
agent. The cytotoxicity assay was performed after 72 h
exposure of the cultured cells to the tested agents at a
concentration ranging from 0.1 to 100 lg cm-3. The SRB
test measuring the cell proliferation inhibition in the in
vitro culture was applied [45]. The cells attached to the
plastic were fixed with cold 50% TCA (trichloroacetic acid,
Aldrich-Chemie, Germany) added on the top of the culture
medium in each well. The plates were incubated at 4 °C for
1 h and then washed five times with tap water. The back-
ground optical density was measured in the wells filled
with culture medium, without the cells. The cellular
material fixed with TCA was stained with 0.4% sulforho-
damine B (SRB, Sigma, Germany) dissolved in 1% acetic
acid (POCh, Gliwice, Poland) for 30 min. The unbound
dye was removed by rinsing four times with 1% acetic
acid, and the protein-bound dye was extracted with 10 mM
unbuffered Tris base (tris(hydroxymethyl)aminomethane,
POCh, Gliwice, Poland) for determination of optical den-
sity (at 540 nm) in a computer-interfaced, 96-well Uniskan
II microtiter plate reader (Labsystems, Helsinki, Finland).
The compounds were tested in triplicate per experiment.
The experiments were repeated at least three times. The
IC50 values were calculated by Cheburator 0.9.0 soft-
ware using a drug–response curve and two-point method
[46].
4-[5-Chloro-2-(2,3,4-trihydroxyphenyl)-1H-benzimidazol-
1-yl]benzoic acid (14, C20H13ClN2O5)
A mixture of 0.50 g 4-[(2-amino-4-chlorophenyl)amino]
benzoic acid (1.9 mmol) and 0.70 g S3TTB (2.1 mmol) in
9.5 cm3 MeOH was refluxed for 4 h. The hot mixture was
filtered and the filtrate was concentrated. Recrystallization
from methanol afforded 0.50 g (66%) 14. M.p.: 194–
ꢀ
197 °C; IR (KBr): m = 3,390 (OH of CO2H), 3,242 (OH),
1,678 (C=O), 1,620 (C=N), 1,501 (C=C), 1,465, 1,306,
1,218 (C–OH), 1,183, 1,142, 1,079, 1056, 994, 993, 922,
861, 800, 755, 713 cm-1; 1H : NMR (500 MHz, DMSO-d6):
d = 8.96 (s, OH), 7.86 (dd, J = 7.9, 1.6 Hz, 1H, Ar–H),
7.30 (m, 2H, Ar–H), 7.05 (d, J = 8.4 Hz, 1H, Ar–H), 6.87
(d, J = 2.4 Hz, 1H, Ar–H), 6.70 (m, 2H, Ar–H), 6.61 (dd,
J = 8.3, 2.2 Hz, 1H, Ar–H), 6.57 (d, J = 8.4 Hz, 1H,
Ar–H) ppm; 13C NMR (125 MHz, DMSO-d6): d = 173.0
(C=O), 168.3, 153.8, 150.7, 149.6, 137.5, 136.8, 136.3,
135.5, 134.5, 133.3, 131.8, 129.5, 129.1, 123.6, 117.8,
116.4, 111.3, 110.4, 104.7 ppm; MS (70 eV): m/z = 396
(M?, 27), 394 (78), 333 (15), 321 (7), 293 (21), 262 (53),
244 (100), 209 (64), 181 (12), 154 (14), 122 (9), 99 (3), 77
(10), 63 (5), 51 (5), 36 (14); HPLC (C-18): log k = 0.026.
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